入侵害虫甘薯凹胫跳甲的鉴定及线粒体基因组分析

Identification and mitochondrial genome analysis of the sweetpotato flea beetle,Chaetocnema confifinis(Coleoptera:Chrysomelidae),an invasive pest in the Chinese mainland

【目的】基于形态学鉴定和分子生物学技术确认甘薯凹胫跳甲Chaetocnema confinis是否入侵中国大陆,测定甘薯凹胫跳甲线粒体基因组序列,分析基因组结构及其系统发育关系。【方法】应用显微镜观察从广东不同地点采集的甘薯凹胫跳甲成虫的形态特征,并扩增cox1基因DNA序列进行分子鉴定;利用Illumina MiSeq测序平台对甘薯凹胫跳甲线粒体基因组进行测序、拼装、注释和特征分析;基于亲缘关系相近种属的线粒体基因组序列进行共线性分析和构建系统发育树,分析基因重排和系统发育关系。【结果】形态和分子鉴定结果表明大陆甘薯上发现的跳甲为甘薯凹胫跳甲。甘薯凹胫跳甲线粒体基因组序列大小为15685 bp,包括有13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码控制区;这37个基因之间排列紧凑,间隔总长度101 bp,排列顺序与模式昆虫Drosophila yakuba线粒体基因排列顺序相同。甘薯凹胫跳甲线粒体基因组A+T含量为77.3%,具有明显的AT偏向性。13个蛋白质编码基因的起始密码子均为ATN。在22个tRNA基因中除trnS1的DHU臂缺失,trnD,trnG,trnN和trnT的二级结构中缺少TψC环外,其余17个都能形成典型的三叶草式二级结构,另trnK的反密码子突变为UUU,trnS1的反密码子突变为UCU。甘薯凹胫跳甲的控制区片段长度仅有60 bp,是目前已报道的昆虫线粒体基因组中最短的控制区。基于线粒体基因组的系统发育分析表明,甘薯凹胫跳甲与跳甲亚科(Alticinae)黄曲条跳甲Phyllotreta striolata亲缘关系最近。【结论】甘薯凹胫跳甲已经入侵到中国大陆。本研究获得了甘薯凹胫跳甲的线粒体基因组序列,为防控甘薯凹胫跳甲和分析叶甲科(Chrysomelidae)各种属间的系统发育关系奠定了基础。

【Aim】The aim of this study is to ascertain whether the sweetpotato flea beetle,Chaetocnema confinis has invaded the Chinese mainland based on morphological identification and molecular biology technique,determine its mitochondrial genome sequence,and analyze its genome structure and phylogenetic relationship.【Methods】The morphological characteristics of C.confinis adults collected from different localities in Guangdong were observed using microscope,and the DNA sequence of cox1 gene was amplified for molecular identification.The mitochondrial genome of C.confinis was sequenced using the Illumina MiSeq sequencing platform,and its sequence was subsequently assembled,annotated and characterized.Collinear analysis was conducted and phylogenetic tree was constructed based on the mitochondrial genome sequences of closely related species to analyze the gene rearrangement and phylogenetic relationship.【Results】The morphological and molecular identification results indicated that the flea beetle on sweetpota found in the Chinese mainland is the sweetpotato flea beetle,C.confinis.The mitochondrial genome of C.confinis is 15685 bp in size,with 13 protein-coding genes,two rRNA genes,22 tRNA genes,and one non-coding control region.All the 37 genes are in compact arrangements with a total interval of 101 bp,the order of gene arrangement is consistent with that in the model insect Drosophila yakuba.The A+T content of the mitochondrial genome of C.confinis is 773%,showing obvious AT bias.The start codons of the 13 protein-coding genes are all ATNs.Except for trnS1 without DHU arm and trnD,trnG,trnN and trnT without TψC rings,a typical cloverleaf-shaped secondary structure can be formed in the remaining 17 tRNA genes,and the mutated anticodons of trnK and trnS1 are UUU and UCU,respectively.The control region fragment of C.confinis is only 60 bp in length,and is the shortest control region in the reported insect mitochondrial genomes.Based on the phylogenetic analysis,C.confinis is closely related to Phyllotreta striolata of Alticinae.【Conclusion】C.confinis has invaded the Chinese mainland.The mitochondrial genome sequence of C.confinis has been obtained in this study,which provides a basis for management of C.confinis and phylogenetic analysis of divergent genera in Chrysomelidae.